Abstract

It has been hypothesized that carotenoid-based sexual ornamentation signals male fertility and sperm competitive ability as both ornamentation and sperm traits may be co-affected by oxidative stress, resulting in positive covariation (the ‘redox-based phenotype-linked fertility hypothesis’; redox-based PLFH). On the other hand, the ‘sperm competition theory’ (SCT) predicts a trade-off between precopulatory and postcopulatory traits. Here, we manipulate oxidative status (using diquat dibromide) and carotenoid availability in adult zebra finch (Taeniopygia guttata) males in order to test whether carotenoid-based beak ornamentation signals, or is traded off against, sperm resistance to oxidative challenge. Initial beak colouration, but not its change during the experiment, was associated with effect of oxidative challenge on sperm velocity, such that more intense colouration predicted an increase in sperm velocity under control conditions but a decline under oxidative challenge. This suggests a long-term trade-off between ornament expression and sperm resistance to oxidative challenge. Shortening of the sperm midpiece following oxidative challenge further suggests that redox homeostasis may constrain sperm morphometry. Carotenoid supplementation resulted in fewer sperm abnormalities but had no effect on other sperm traits. Overall, our data challenge the redox-based PLFH, partially support the SCT and highlight the importance of carotenoids for normal sperm morphology.

1. Introduction

Female preference for a particular male phenotype has long been recognized as a potent selective pressure driving the evolution of conspicuous secondary sexual characters such as carotenoid-based ornamentation [1,2]. The level of expression of such characters is known to be a major determinant of male mating opportunities, and thus reproductive success [2]. In addition, sexual selection continues after copulation in species where females copulate with multiple males, leading to competition between male spermatozoa for the fertilization of female ova [3]. In such species, male reproductive success is a result of an interplay between the precopulatory and postcopulatory phases of sexual selection [4]. Despite many recent studies focusing on interaction between these phases, there have been no conclusive results explaining how the two processes integrate, with a positive relationship being observed in some studies [5–10] and a negative relationship [11–16], or no relationship, in others [17–19].

A positive relationship is predicted by the ‘phenotype-linked fertility hypothesis’ (PLFH), which proposes that male fertility is signalled through secondary sexual characters [20]. The hypothesis proposes that both sexual signals and sperm traits are phenotypically plastic and co-affected by environmental effects, resulting in a positive correlation between signal expression and functional fertility [20]. As both sexual signals [21] and spermatozoa [22] are believed to be sensitive to oxidative stress, Blount et al. [23] hypothesized that oxidative stress could be the major factor linking such environmental effects to ornamentation and functional fertility (hereinafter ‘redox-based PLFH’).

Oxidative stress is characterized by an accumulation of oxidative damage to cellular components (e.g. DNA, lipids, proteins) resulting from oxidation by free radicals and other reactive oxygen and nitrogen species (ROS), and can be prevented by antioxidant mechanisms [21]. ROS are inevitable by products of oxidative metabolism and their formation generally increases under metabolically demanding and stressful conditions or during an immune response [21,24,25]. To ensure high motility, spermatozoa are metabolically active and rich in polyunsaturated fatty acids that oxidize more readily than monounsaturated and saturated fatty acids [22]. Moreover, spermatozoa may lack the capability to repair DNA as transcription and translation probably stop after spermiogenesis [26], and most enzymatic and non-enzymatic antioxidants are lost with the cytoplasm during sperm elongation [27]. Taken together, these factors render spermatozoa highly sensitive to oxidative stress, which is considered a major cause of male infertility in humans [22].

According to the redox-based PLFH [23], males that are more colourful should have a high-quality antioxidant system that better protects their sperm against oxidative stress. By choosing more ornamented males, therefore, females would obtain a direct benefit through fertility insurance, as well as an indirect benefit in the form of superior DNA integrity for their offspring.

An alternative theory, the ‘sperm competition theory’ (SCT), predicts a negative relationship between precopulatory and postcopulatory sexual traits based on the assumption that the amount of resources that can be invested in reproduction is limited [4]. As a result, a trade-off is expected between investment in precopulatory and postcopulatory traits. Based on this theory, we propose that expression of carotenoid-based ornamentation may be traded off against sperm resistance to oxidative challenge.

It has been proposed that the direction of any relationship between traits traded off against each other will depend on the amount of resources available to the individual [28]. Hence, a positive correlation may be observed under favourable conditions when resources are abundant, while a negative correlation may occur under more stressful conditions when resources are limited. Experimental manipulation of stress levels or resource availability are essential, therefore, to reveal trade-offs that might be masked by variation in these factors in observational studies [28–30].

In species with carotenoid-based ornamentation, carotenoids may be the key resource traded off between ornamentation and sperm traits. Particularly so given their antioxidant properties [21] and reported beneficial effects on sperm quality and male fertilization success in many taxa, including humans [7,23,31]. In birds, however, the importance of carotenoids for both antioxidant protection [32,33] and sperm competitive ability is disputed [10,31,34,35], and a pro-oxidant effect of high carotenoid concentrations has even been propounded [36]. Further studies are needed, therefore, to elucidate the importance of carotenoids as regards spermatogenesis and sperm competition in birds.

Both the redox-based PLFH and the SCT assume there is an environmental component of variation in both sexual signalling and sperm traits. In animals, sperm traits have indeed been found to be affected by environmental factors known to influence individual oxidative status, such as temperature, workload, parasites, immune activation and social environment (e.g. [37–40], reviewed in [41]). While a negative correlation between sperm oxidative damage and sperm velocity/viability has been documented in the wild [10,39], oxidative stress as a causal factor in such environmental effects cannot be inferred unless such studies use some form of controlled oxidative challenge.

In this study, we manipulate oxidative status and carotenoid availability in adult zebra finch (Taeniopygia guttata) males to test their effect on sperm traits and the relationship between sperm traits and carotenoid-based sexual ornamentation. The birds were subjected to controlled oxidative challenge induced by diquat dibromide (hereinafter referred to as diquat), a redox-cycling agent known to produce superoxide radicals in vivo that has recently been recognized as a convenient oxidative stress inducer in both ecological studies [42–45] and laboratory models of Parkinson's disease [46]. In order to assess changes in sperm quality, we analysed several sperm traits that have previously been suggested as important for male fertility and sperm competitive ability, i.e. sperm velocity [47–49], total sperm length (TSL) [50–52], relative flagellum length [50], relative midpiece length [50,53] and abnormal sperm proportion [54]. These traits could potentially be affected by oxidative stress since sperm development, as well as sperm motility, are dependent on sperm cell membrane fatty acid composition, which can be altered under oxidative stress [27,55]. Both the mitochondria-containing sperm midpiece and sperm velocity could further be affected by oxidative stress through its effect on energy and ROS production in mitochondria [56].

Experimental oxidative challenge is expected to divert resources to antioxidant defence, thereby reducing investment in sexual traits and revealing a possible trade-off [28,29]. As TSL, relative midpiece and flagellum length and sperm velocity are usually considered to be positively associated with sperm competitive ability [47,49–52], we predicted oxidative challenge-induced changes in their expression to be positively correlated with ornament expression under the redox-based PLFH, and negatively correlated under the SCT. The opposite is predicted for abnormal sperm proportion, as high occurrence of sperm abnormalities reduces male fertility [54]. In addition, we predict that carotenoids will counteract the potential effects of oxidative challenge on both sperm traits and ornamentation–sperm trait relationships if carotenoids act as antioxidants in the testes or ejaculate, or amplify such effects if they act as pro-oxidants.

2. Material and methods

(a) Study species and housing

This experiment was carried out on 60 adult zebra finch males at an indoor facility of the Institute for Vertebrate Biology (Studenec, Czech Republic) from July to August 2011. All birds were 1–1.5 years old at the time of the experiment and were housed individually in 60 × 40 × 40 cm cages with millet seed, cuttlefish bone, grit with crushed shell and water provided ad libitum. Four weeks before the start of the experiment, the millet seed was replaced with hulled millet seed, which was used for carotenoid supplementation during the experiment. To stimulate breeding condition in males, the original 10 : 14 (light : dark) winter photoperiod was gradually changed to 14 : 10 over April and May. Male breeding condition was further stimulated by placing eight females in separate cages in close proximity to those of the males at the end of May, thereby providing both visual and vocal contact. No further changes in social environment were made thereafter.

This same experiment was also used for testing the antioxidant function of carotenoids in vivo and the effect of oxidative challenge on carotenoid-based ornamentation and circulating carotenoid levels [45]. Here, we re-use the data on beak colouration for testing a different set of hypotheses.

(b) Experimental design and sample collection

Males were randomly assigned to four treatment groups with 15 individuals in each. For 70 days, we experimentally manipulated the level of oxidative burden and carotenoid intake in a 2 × 2 factorial design such that each treatment group received one of the four possible combinations of low (ROS−) or high (ROS+) oxidative burden and low (CAR−) or high (CAR+) carotenoid intake.

Diquat dibromide (Reglone 200 g l−1, Syngenta, UK), a chemical known to generate superoxide anions in vivo [57], was used for controlled oxidative challenge (ROS+ groups). In order to imitate a long-term mild increase in oxidative burden, diquat was administered in drinking water at a sublethal dose of 25 mg l−1 (approx. 3–6 µg g−1 of body weight), which has been shown to have no long-term effect on the bird's clinical condition [45]. The control groups (ROS−) received plain drinking water instead. Thirteen diquat-treated birds showed mild apathy and ruffled feathers at the beginning of the treatment (Yates' χ2-tests: ROS effect, χ2 = 14.14, p < 0.001; carotenoid effect, χ2 = 0, p = 1). Aside from two birds from the ROS+CAR− group and two birds from the ROS+CAR+ group that died, the birds recovered after a few days. There was no significant difference in mortality between diquat- or carotenoid-treated birds and the control birds (Yates' χ2-tests: ROS, χ2 = 2.41, p = 0.12; CAR, χ2 = 0.27, p = 0.61).

For the high carotenoid intake diet (CAR+), 200 mg of lutein and zeaxanthin (1 ml of FloraGLO Lutein 20% SAF, Kemin/DSM, France) and 1 ml of safflower oil (Jules Brochenin, France) were mixed with 1 kg of hulled millet seed. This concentration, which is close to the upper limit that zebra finches can assimilate [58], was chosen as it is most likely to reveal the hypothesized pro-oxidant effect of carotenoids. Given that assimilation of carotenoids reaches a plateau at between 100 and 200 µg g−1, and that wild songbirds are known to receive up to 100 µg g−1 carotenoids in their food [59], we argue that such a dose results in plasma concentrations that are within the range songbirds may experience in the wild (see also [45]). The control diet (CAR−) was prepared by mixing 1 kg of seed with 2 ml of safflower oil and 10 mg of α-tocopherol (T3251, Sigma-Aldrich, Czech Republic) to compensate for the safflower oil and α-tocopherol contained in the FloraGLO. Both prepared diets were frozen at −80°C until use to prevent carotenoid degradation.

Beak colour measurements and ejaculate samples were collected at both the beginning and end of the experiment. All measurements in this study were undertaken blind with respect to sample identity and experimental treatment.

(c) Beak colouration

Beak reflectance was measured between 300 and 700 nm using an AvaSpec 2048 spectrophotometer with an AvaLight-XE light source (Avantes, The Netherlands). Four points on the upper beak and two on the lower beak were measured on each side with the probe held perpendicular to the surface. The spectrophotometer was standardized against a darkroom and WS-2 white standard after measuring five individuals. Hue, red chroma and UV chroma were calculated from the reflectance data using the ‘pavo’ package [60] in R v. 3.0.2 (the R Foundation for Statistical Computing, Austria; for details see [45]). Because both hue and logit transformed UV chroma were strongly correlated with red chroma (Spearman's rs = 0.76 and rs = −0.88, respectively), but did not meet the assumption of normality (Shapiro–Wilk's test: W = 0.958, p = 0.039 and W = 0.862, p < 0.001, respectively), red chroma was used as a representative variable throughout the study.

In those cases where beak red chroma was significantly correlated with sperm traits, we calculated whether sperm trait differences could be discriminated based on beak colouration by zebra finch eyes using the colour opponency model [61,62] implemented in the ‘pavo’ package [60]. To this end, males were divided into three groups of 20 individuals according to the sperm trait tested (e.g. short, medium and long sperm) and the reflectance spectra in each group were averaged. Colour contrasts (ΔS) were subsequently calculated between each group pair based on neural noise [61,62], using characteristics of the zebra finch visual system [63,64]. The Weber fraction was set at 0.05 [62] and standard daylight D65 was used as the illuminant. Just noticeable differences (jnds) were used to measure ΔS, with values of ΔS > 1 jnd theoretically discernible by the zebra finch visual system under the above conditions.

(d) Sperm measurement

Ejaculate samples (approx. 0.5 µl) were obtained by gentle cloacal massage [65] and immediately diluted in 60 µl of Dulbecco's modified Eagle’s medium (DMEM; Invitrogen, USA). Approximately 3 µl of the diluted sample was subsequently used for analysis of sperm velocity, the rest being stored in 10% formalin for analysis of sperm morphometry and abnormal sperm proportion.

Immediately after sample collection and dilution, the sample was placed on a Leja slide (Leja, The Netherlands) and sperm movement was recorded with an Olympus CX41 light microscope equipped with a heating table, phase contrast and a digital UI-1540-C camera (Imaging Development Systems, Germany). All recordings were performed at 40°C. The recordings were later analysed using the CEROS computer-assisted sperm analysis system (Hamilton Thorne, USA). As the three sperm velocity characteristics (straight, curvilinear and average path velocity) were highly inter-correlated, we used curvilinear velocity (VCL) only for all further analysis [65].

Sperm samples stored in formalin were smeared onto glass slides, left to dry and then photographed using an Olympus BX51 light microscope equipped with a camera (Visitron Systems, Germany). Sperm morphometry was subsequently measured using Olympus QuickPHOTO Industrial 2.3 imaging software. For each sample, we measured the length of the head, midpiece and tail and calculated TSL (the sum of the three components) on 30 morphologically normal spermatozoa [65]. All morphometric measurements were undertaken by the same person (M.N.).

The proportion of morphologically normal and abnormal spermatozoa in each sperm sample was assessed at 400 times magnification, with 100 sperm cells analysed per sample for each bird. Sperm not showing the typical songbird helical sperm head-shape were considered abnormal, as were the few sperm cells showing tail deformities (two-tailed spermatozoa with one head and one midpiece) [65]. All scoring was undertaken by the same person (P.O.).

(e) Statistical analysis

All data analysis was carried out using R v. 3.3.0 (the R Foundation for Statistical Computing, Austria). We first explored Pearson's correlations between individual sperm traits and ornament expression, with proportional variables (red chroma, flagellum/TSL proportion, midpiece/flagellum proportion and proportion of sperm abnormalities) normalized by logit transformation using the ‘car’ package. Subsequently, effects of experimental manipulation were analysed using linear models, with change scores of each sperm trait set as dependent variables. Oxidative challenge and carotenoid intake (both control and high), together with their interaction, were included as factors in order to test their effect on sperm traits. To control for the effect of initial sperm trait expression on its subsequent change, we also included pre-experimental values of the sperm trait analysed, together with its two-way interaction with both treatment factors. To test whether ornament expression signals, or is traded off against, sperm resistance to oxidative challenge, pre-experimental values for beak red chroma, along with its interaction with oxidative challenge, were inserted into the models. To test whether initial ornament expression predicts sensitivity of the sperm traits to carotenoid intake, the interaction between initial colouration and carotenoid intake was also included. Real-time trade-off in allocation of available resources between ornament expression and sperm traits was tested by including the change in beak red chroma and its two-way interaction with each of the treatment factors (see electronic supplementary material, tables S5 and S6 for a summary of all predictor terms included in each global model). Global models were simplified by removing all non-significant (i.e. p > 0.05) terms using a stepwise procedure. In the final models, all predictor variables were centred to obtain biologically relevant main effects in the presence of interactions [66].

Neither TSL nor relative flagellum length (flagellum/TSL ratio) was correlated with sperm velocity, though there was a marginally non-significant positive correlation between sperm velocity and relative midpiece length (electronic supplementary material, table S2). Interestingly, a longer midpiece was also associated with a lower proportion of sperm abnormalities. The relationship between relative midpiece length and sperm velocity was not due to differences in abnormal sperm proportion as sperm velocity and abnormal sperm proportion were uncorrelated. Descriptive statistics for sperm trait measurements are provided in electronic supplementary material, tables S3 and S4.

Contrary to the predictions of the redox-based PLFH, a more intense initial beak colouration was associated with a decrease in sperm velocity under oxidative challenge, while a positive correlation was observed under control conditions (table 1, figure 1; electronic supplementary material, table S5). The observed change in sperm velocity, however, was not related to change in beak colouration or its interaction with oxidative challenge. Further, neither sperm velocity nor the effect of oxidative challenge on this trait was influenced by carotenoid intake.

Effects of oxidative challenge, carotenoid intake and ornament expression on sperm traits. Estimates are coefficients from minimal adequate models with controls and elevated treatment levels coded 0 and 1, respectively. All predictor variables were centred to enable interpretation of the main effects with interactions present. For full details of the models and the selection procedures used, see the Statistical analysis section. Full models are available in the electronic supplementary material. TSL, total sperm length.

Oxidative challenge also resulted in spermatozoa with a lower midpiece/flagellum ratio (table 1; electronic supplementary material, table S6). This effect was relatively weak, however, and was driven mainly by midpiece shortening, as there was no effect of oxidative challenge on TSL or flagellum/TSL ratio. Carotenoid intake had no effect on any of the morphometric traits. Interestingly, changes in both TSL and flagellum/TSL ratio were negatively correlated with initial beak colouration, irrespective of treatment.

Oxidative challenge did not affect abnormal sperm proportion in our experiment (table 1; electronic supplementary material, table S6). On the other hand, abnormal sperm proportion was reduced by elevated carotenoid intake. The significant interaction of carotenoid intake with initial abnormal sperm proportion further indicated that high carotenoid intake reduced the occurrence of sperm abnormalities most strongly in males having the highest proportion of abnormal sperm prior to the experiment (figure 2). This effect was even more significant after removing one influential observation (Cook's distance > 1; electronic supplementary material, table S7).

Effect of carotenoid availability on change in abnormal sperm proportion. Lines are predicted values from the minimal adequate model.

4. Discussion

In our study, we found little support for the PLFH [20], and especially for the redox-based variant, which proposes that high-quality carotenoid-based ornamentation signals high sperm resistance to oxidative challenge [23]. First, prior to the experiment, carotenoid-based beak colouration was positively correlated with sperm length and relative flagellum length only, but not with sperm velocity, relative midpiece length or abnormal sperm proportion. Sperm length has recently been reported as predicting fertilization success in zebra finches [52], though this was probably related to higher sperm velocity in males selected for longer spermatozoa [52,67]. In our study, however, sperm length was not correlated with sperm velocity and there was no relationship between sperm velocity and ornament expression. Second, we observed a decrease in sperm length in more colourful males (pre-experiment) during the course of the experiment, irrespective of treatment. This further challenges interpretation of a positive correlation between initial beak colouration and sperm length as supporting the PLFH. Third, initial beak colouration predicted the effect of oxidative status on sperm velocity, such that more intense beak colouration predicted an increase in sperm velocity under control conditions and a decline in sperm velocity following diquat exposure. Since the primary mechanism of diquat toxicity is induction of oxidative stress through a redox-cycling reaction generating superoxide anions [57], these results suggest that more ornamented males have sperm that are less resistant to oxidative challenge. Such a result is inconsistent with the redox-based PLFH [23]; rather, it suggests a trade-off between carotenoid-based ornamentation and sperm resistance to oxidative challenge.

No loss in body mass was observed in the diquat-exposed birds in our study, indicating that the diquat-induced effects were not a consequence of reduced food intake or impaired total intestinal nutrient absorption (see also [45]). Using this same experiment, we previously reported that blood oxidative damage remained unchanged under diquat exposure, but that blood antioxidant capacity increased markedly [45]. This suggests that antioxidant response, rather than elevated oxidative damage, plays a major role in the diquat-induced effects observed.

A trade-off between investment in precopulatory and postcopulatory traits is predicted by the SCT, which assumes that resources available for allocation to either trait type are limited [4]. Interestingly, the change in sperm velocity noted was not related to change in beak colouration, suggesting that there is no direct trade-off in allocation of available resources between these two traits. Instead, the adverse effect of oxidative challenge on sperm velocity was related to high initial beak colouration, which suggests a long-term trade-off between ornament expression and sperm resistance to oxidative challenge. This could imply a long-term negative effect of investment in ornamentation on oxidative sperm resistance. An alternative, though not mutually exclusive, explanation may reside in differences in individual life histories. Accumulating evidence suggests that individuals are adapted for specific environments, with conditions experienced in early development shaping an individual's phenotype for the rest of its life [68]. It has been shown that early-life stress may result in lower investment in sexual trait expression when adult [69–71] and that a mismatch between early-life and adult environments may have deleterious consequences for individual fitness [72]. Accordingly, those males with redder beaks in our study may be adapted to low-stress environments and have limited antioxidant mechanisms. As a result, they may suffer a decline in sperm function under oxidative challenge. By contrast, males with paler beaks may be better adapted to higher stress environments and, therefore, perform better under oxidative challenge. The potential long-term carry-over effects of investment into sexual ornamentation on sperm traits, as well as life-history-related physiological constraints and differences in trade-off solving are not usually considered in studies investigating the relationship between precopulatory and postcopulatory sexual traits. As such, these should provide interesting areas for future research.

Our results, showing lower sperm resistance to oxidative challenge in more colourful males, contrast with those of a previous study reporting higher resistance to the adverse effects of experimental brood enlargement on sperm motility, velocity and oxidative damage in more colourful great tit (Parus major) males [10]. A possible explanation for such a discrepancy could lie in a difference in the signalling content of pigmented bare body parts and plumage. This is supported by their differing responses to oxidative challenge, with reduced colour intensity observed in bare-part ornamentation [44,45] but not in plumage ornamentation [73,74] following oxidative challenge. Alternatively, the contrasting results may reflect interspecific differences in ornament production and function, or differences in methodology (e.g. other effects, unrelated to oxidative stress, may come into play when using brood enlargement [10]). Whatever the explanation, our data demonstrate that carotenoid-based condition-dependent sexual traits do not generally signal sperm quality, as predicted by the PLFH [20,23].

Oxidative challenge also resulted in a shortening of sperm midpiece length in our study. Traditionally, sperm morphometry was thought to be genetically determined, at least in internal fertilizers, with low intra-individual plasticity [75,76]. Despite this, effects of season, temperature and social environment on sperm morphometry have been reported in a number of studies [38,77,78]. Our results, showing shortening of midpiece length following oxidative challenge, corroborate plasticity in morphometric sperm traits in response to environmental factors and suggest that it may be mediated by oxidative stress.

The sperm midpiece contains mitochondria that produce energy for sperm motility, and the length of the midpiece has previously been shown to be positively correlated with sperm velocity, both at the intraspecific [53] and interspecific [50] levels. In our study, however, we observed a weak and marginally non-significant correlation between midpiece length and sperm velocity, while the effects of oxidative challenge on midpiece length and sperm velocity differed, suggesting that midpiece length is probably not the major determinant of sperm velocity.

Oxidative stress is generally considered one of the most important causes of morphological defects in spermatozoa [22]. Surprisingly, we found no effect of oxidative challenge on abnormal sperm proportion in our study. We previously documented an adaptive antioxidant response in this experiment [45], however, and this is likely to have prevented any increase in oxidative damage in the testes, thereby preventing any increase in sperm abnormalities.

Carotenoid supplementation reduced the occurrence of sperm abnormalities, particularly in males with an initially high abnormal sperm proportion, suggesting that the carotenoids used in our study (i.e. lutein and zeaxanthin) are important for normal spermatogenesis. To date, evidence for the importance of carotenoids regarding normal sperm morphology has been mixed, with lycopene most often receiving support ([79–81], but see [82]). A positive correlation between normal sperm morphology and lutein or zeaxanthin concentration has only been reported in one study [83], most other studies reporting no association [81,82,84]. To the best of our knowledge, our study provides the first experimental evidence for the positive effect of lutein or zeaxanthin on normal sperm morphology. As a high occurrence of sperm abnormalities is a known cause of impaired male fertility [54], our data suggest that reduced fertility and sperm competitiveness previously reported in carotenoid deficient males [85] may be due to a high proportion of abnormal sperm in their ejaculates.

The positive effects of carotenoids on sperm traits is usually assumed to be due to their antioxidant properties [23,79–81,83]. In birds, however, the importance of carotenoids as antioxidants is disputed; indeed, carotenoids have even been proposed as pro-oxidants [31,34,36]. In our study, we observed no adverse effect of high carotenoid intake on any sperm trait, which is inconsistent with a pro-oxidant effect. By contrast, the antioxidant function of carotenoids in avian testes was supported by their reducing effect on abnormal sperm proportion. Support for their antioxidant function was mixed, however, as carotenoids did not inhibit the effects of oxidative challenge on sperm velocity or midpiece length. Overall, our results suggest that a positive carotenoid effect on sperm morphology is either not mediated by antioxidant function or that such a function is important only for specific sperm traits.

In conclusion, our data challenge the redox-based PLFH and suggest that expression of carotenoid-based sexual ornamentation is instead traded off against sperm resistance to oxidative stress, as predicted by the SCT. In their review, Dowling & Simmons [86] proposed that redox homeostasis could constitute a major constraint in life-history evolution, possibly underlying negative associations between individual sperm traits (e.g. sperm number and sperm quality) or between sperm traits and immune function. Our results suggest that redox homeostasis may also drive a trade-off between expression of sexual ornamentation and sperm competitive ability. The absence of any real-time trade-off between precopulatory and postcopulatory traits in our data implies that such a trade-off may involve long-term carry-over effects and/or physiological constraints associated with different life histories. In addition, our results suggest that carotenoids are important for normal spermatogenesis, thereby influencing male fertility and sperm competitive ability.

Ethics

All experimental procedures were conducted in accordance with the Guidelines for Animal Care and Treatment of the European Union, and were approved by the animal care and ethics representatives of the Faculty of Science of Charles University in Prague and the Czech Academy of Sciences (no. 041/2011).

Data accessibility

Authors' contributions

T.A. and O.T. conceived and designed the study; O.T. conducted the experiment; O.T., J.A. and T.A. collected the data; O.T. analysed the spectrophotometric ornament data; J.A., M.N. and P.O. analysed sperm data; O.T. carried out the statistical analysis and drafted the manuscript with input from T.A. All co-authors contributed to the final version of the manuscript and gave approval for its publication.

Competing interests

The authors declare no competing interests.

Funding

This study was funded through a Czech Science Foundation grant (no. P506/12/2472). O.T., J.A. and M.N. were supported through an institutional project of Charles University (no. SVV 260313/2016).

Acknowledgements

We thank Barbora Gabrielová, Jana Svobodová and František Vejmělka for help with data collection and Helena Hejlová for help with bird care.

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